Electrophoresis is a generally inexpensive method of focusing analyte molecules, e.g., proteins, based on their movement in an electric field. Electrophoresis can be carried out in free solution or with the aid of a support medium, such as a gel, polymer solution, or granular packing. Electrophoresis typically employs a buffered electrolyte to maintain pH and provide sufficient conductivity to allow the passage of current. Electrophoretic focusing techniques have been described by a well known flux equation, with focusing occurring at the point in the processing chamber where the net gradient vanishes. A method known as counteracting chromatographic electrophoresis (CACE) has been used to focus proteins at the interface between two different gel filtration media packed into the upper and lower halves of an electrochromatography column. Science 1985, 227, 1586-1588. At least one protein, ferritin, could be concentrated beyond 100 mg/mL. Sep. Sci. Technol. 1988, 23, 875; Sep. Purif. Methods 1989, 18,1. This sucees was tempered, however, by the finding that this approach worked poorly with protein mixtures and would be difficult to scale up. Biotechnol. Prog. 1990, 6, 21.
It has been demonstrated that charged proteins can be focused, e.g., separated, concentrated, etc., using an electric field gradient in an electrochromatography column. Koegler and Ivory, J. Chromatogr., A 1996, 229, 229-236. A fluted cooling jacket was used to form a linear gradient in the electric field which drove the proteins against a constant flow of buffer in a packed dialysis tube. This approach was slow and cumbersome and gave mediocre results, but it successfully illustrated a focusing technique known as electric field gradient focusing (EFGF). It also has been shown that proteins would focus in the electric field gradient formed by an axial conductivity gradient and opposed by a constant flow of buffer. Greenlee and Ivory, Biotechnol. Prog. 1998, 14, 300-309. The device was surprisingly fast when run in free solution, reaching equilibrium in less than 10 min., and gave unexpectedly good results when filled with a 40-μm size exclusion (SEC) packing. Focusing can also be achieved by opposing a constant convective velocity with a gradient in the electrophoretic velocity of the protein. This gradient can be created by varying the net charge on the protein (as in isoelectric focusing), by varying the cross-sectional area through which the electric current travels, as with electric field gradient focusing, or by varying the buffer conductivity. Isoelectric focusing (IEF) is a gradient focusing method which varies the charge on a protein using a pH gradient. The convective velocity is usually set to zero while the net charge on the protein decreases as it approaches its isoelectric point (pI). The protein focuses at this point since its net charge, and therefore its electrophoretic velocity, both vanish at its pI.
Conventional IEF is usually performed in a support medium such as agarose or polyacrylamide gel. The pH gradient is formed by using a complex set of reagents known as carrier ampholytcs which generate a stable, linear pII gradient under the influence of an applied electric field. Proteins migrate to the region where the ampholyte solution pH is equal to its own pI. In gels, detection of the focused bands involves a time consuming stain/destain procedure, and the ampholytes should be removed before the stain is applied. Established IEF protocols and a succinct history of its development are given by Righetti (1983).
Certain known electrophoretic systems utilize a confined chamber formed between two plates. An electric field is established between two opposing electrodes or in a desired two-dimensional array using additional electrodes. Such systems typically have good resolution of analytes, e.g., proteins, but have limited flow rates. Thus, electrophoresis has, in general, not been performed at flow rates adequately adapted to preparative scale production to provide significant quantities of a desired product. Filtration and chromatography, the principal methods used for preparative scale purification of biopharmaceuticals, have shortcomings, such as difficulty separating isoforms.
An electrophoretic method and apparatus for focusing solutes is described in U.S. Pat. No. 6,277,258 to Ivory et al., which is commonly assigned with the invention disclosed here and is hereby incorporated by reference in its entirety for all purposes. The disclosed apparatus utilizes a dynamic field gradient focusing (DFGF) method wherein dynamic electric field gradients are created, for example by a computer-controlled external circuit which manipulates the field strength generated by an array of electrodes. Dynamic field gradient focusing is well adapted to concentrate a target analyte or species from a dilute fluid sample, in certain cases being able to separate one or more such analytes from other species present in the fluid sample by concentrating them at different locations in the DFGF chamber. Such electrophoretic focusing takes advantage of the target analyte's charge to mass ratio or electrophoretic mobility as the fluid carrying the analyte is passed through an electric field gradient in a DFGF chamber. An electrophoretic approach such as DFGF can exploit small differences in net charge on an analyte molecule to separate isoforms by their electrophoretic mobilities. DFGF can be used to focus certain charged analytes, for example a desired protein, either on a batch-by-batch basis or in a continuous fashion. In addition, DFGF can be used in some cases to separate a desired analyte from other analytes in the same fluid sample, by concentrating the desired analyte at a location in the DFGF chamber at which the other analytes do not concentrate under the conditions used. The operative conditions typically include the sample flow rate, field strength and gradient, properties of the carrier fluid such as pH, chamber and electrode configuration, etc.
Electrophoretic devices and methods also are disclosed in U.S. Pat. No. 5,298,143 to Ivory et al., which is commonly assigned with the invention disclosed here and is incorporated herein by reference in its entirety for all purposes. Separation is performed, optionally with an electrode array, in an annular chamber formed between a fixed surface and a rotating surface. A disadvantage of the DFGF methods and devices disclosed by the Ivory et al. patents and others is that relatively small quantities of the desired concentrated product might be produced over a given period. Thus, for example, dynamic field gradient focusing was demonstrated to separate proteins by their electrophoretic mobility at the analytical scale in a device limited to microgram loads of protein (2.5 PE/PC, 10 Myo) using a small packed column to separate a cocktail of three proteins: phycoerythrin, phycocyanin, and myoglobin into two visible bands. Huang and Ivory, Anal. Chem. 1999.
Improvements are needed to increase the usefulness of electrophoretic processing of analytes. Notably, for example, improvements of known electrophoretic techniques are needed to scale-up DFGF for larger analyte loads. Scaling requires overcoming challenges in the areas of natural convection of the analyte in the processing chamber, cooling, and electrode arrangement. Natural convection will counteract focusing forces. Cooling, primarily dissipation of Joule heat, is important for certain analytes, such as proteins, since elevated temperatures can denature proteins.
Thus, despite advances in the electrophoretic methods and devices noted above, a need exists for electrophoretic methods and devices that can effectively focus analytes, e.g., separate charged solutes, such as protein mixtures, into their component solutes. The present invention seeks to fulfill these needs and provides further related advantages. Thus, it is an object of the present invention to address one or more of the above-mentioned research and industrial needs. It is an object of certain, but not necessarily all, exemplary embodiments of the invention to provide devices and methods for processing analytes, especially for focusing charged analytes, i.e., for concentrating and/or separating such charged analytes, e.g., separating charged analytes from other species in a fluid sample. From the following summary and the detailed description of certain exemplary embodiments, additional objects of the invention and objects of certain exemplary embodiments of the invention will be apparent to those skilled in the art, i.e., to those having skill and experience in this area of technology.